Concurrent Inhibition of Beta-Catenin/CBP and FLT3 Strongly Suppresses c-Myc and Synergistically Induces Apoptosis in FLT3 Mutant AML Cells

Introduction. The FLT3-ITD mutations are frequently observed in acute myeloid leukemia (AML) and correlate with poor prognosis. FLT3-ITD signaling activates MAPK/ERK, JAK/STAT5, and PI3K/AKT pathways, and stabilizes and increases the nuclear localization and transcriptional activity of β-catenin. The effectiveness of FLT3 tyrosine kinase inhibitors (TKIs) is often limited because of additional mutations in the FLT3 gene, TKI-induced alternative signaling activation, and lack of TKIs against AML stem cells (LSCs). Interestingly, the novel β-catenin/cAMP response-element binding protein (CBP) modulator C82 binds to CBP and inhibits its interaction with β-catenin, and disrupts Wnt/β-catenin mediated cell proliferation, self-renewal and survival, which are essential for LSCs/progenitor cells. Consequently, we evaluated the effects of C82 in combination with TKIs in AML cells with FLT3 mutations.

Methods. BaF3 cells harboring various FLT3 mutations, Molm13 and MV4-11 cells with FLT3-ITD mutation, as well as FLT3-wild type BaF3 and OCI-AML3 cells were treated with C82 or TKI (sorafenib or quizartinib) alone, or in combination. Apoptosis and cell cycle distribution were determined by flow cytometry, synergistic cytotoxicity (CI) of the combination treatment was calculated by isobologram analysis, and cellular localization of β-catenin was determined by confocal imaging. Protein expression was measured by western blotting and RNA expression by qRT-PCR.

Results. Combination of C82 with sorafenib synergistically induced apoptosis in BaF3 FLT3-ITD, BaF3 FLT3-D835G, BaF3 FLT3-ITD plus 676, Molm13, and MV4-11 cells, and in sorafenib resistant BaF3 FLT3-ITD plus 842 cells, even when they were co-cultured with human bone marrow-derived mesenchymal stem cells (MSCs) to mimic the AML microenvironment (CI < 1). Similar results were obtained with Molm13 and MV4-11 cells treated with C82 plus quizartinib. Cell cycle analysis showed that the combination of C82 and sorafenib suppressed cell cycle progression and increased the proportion of cells in subG₁ and G₁ phases. Furthermore, the combination of C82 and sorafenib inhibited nuclear localization of β-catenin and reduced CD44 levels. Pathway PCR array analysis revealed that C82 and sorafenib, either alone or in combination, suppressed the expression of survivin, cyclin D1, and c-Myc, all downstream targets of β-catenin and several frizzled family receptors/co-receptor and ligands, such as FZD1, FZD3, FZD5, FZD6, FZD7, low-density lipoprotein receptor, WNT10B, and WNT3 (≥2 fold). Western blot analysis confirmed that C82 and TKI (sorafenib or quizatinib) either alone or in combination suppressed c-Myc, survivin, and CD44. Selective inhibition of FLT3-ITD downstream signaling indicated that TKI-induced c-Myc inhibition is primarily mediated through the FLT3-ITD/STAT5/c-Myc signaling cascade.

Conclusion: These results demonstrate that combined inhibition of β-catenin and FLT3 tyrosine kinase exerts synergistic cytotoxicity in AML cells with FLT3 mutations. These findings provide a rationale for developing clinical protocols employing β-catenin/CBP modulators and TKIs to overcome FLT3 inhibitor resistance and to determine whether treatment outcomes in patients with FLT3 mutant AML can be improved.